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Background: Oxidative stress is an invasive condition with increased reactive oxygen species, now recognized as an important characteristic of malignant disorders as well as their progression. Aims: The aim of this study was to evaluate the role of oxidative stress induced genes and antioxidative enzymes in myeloproliferative neoplasms (MPN): polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Methods: Using microarray analysis we studied oxidative stress induced gene expression in CD34+ hematopoietic progenitors of MPN patients. An assay for superoxide dismutases (SOD) was based on the ability of SOD to inhibit the autoxidation of epinephrine at alkaline pH. The activity of glutathione reductase (GR) was based on the capacity of GR to catalyze the reduction of oxidized to reduced glutathione using NADPH as a substrate. Glutathione peroxidase activity was assayed following the oxidation of NADPH with t-butylhydroperoxide as a substrate. The antioxidative enzymes activities were determined in red blood cells lyzate. Results: Oxidative stress induced FBJ murine osteosarcoma viral oncogene homolog (FOS) gene expression was highly elevated in ET (3.1 fold) and PV (3.7 fold) comparing to healthy controls. FOS gene expression was higher in JAK2V617F heterozygous PV patients (4.1 fold). Less prominent expression was observed for kelch-like ECH-associated protein 1 (KEAP1) gene in PV (1.6 fold) and PMF (1.8 fold). Regarding ET patients, heme oxygenase 1 (HMOX1) gene was preferentially expressed in JAK2V617F positive ET (2.4 fold), significantly higher than in healthy controls (p

Purpose: The purpose of this study was to examine the gene expression profile of granulocyte colony stimulating factor (G-CSF)-mobilized peripheral blood (mPB)-derived progenitors, used in transplantation. Methods: We correlated gene expression patterns of highly enriched steady-state peripheral blood (PB)- and mPB-derived CD71(+) cells by microarray and ingenuity pathway analyses, to identify the transcriptional program during in vitro erythroid differentiation. Results: The gene expression was more than doubled in mPB-derived (4180 genes) compared to PB-derived erythroid progenitors (1667 genes) while PB-and mPB-derived erythroid progenitors shared 1534 common genes. Comparative analysis of transcript levels showed differential expression of 54 genes between cultured erythroid progenitors of PB and mPB origin, where we identified common 13 downregulated and 30 upregulated genes. The most significant genes in mPB-derived erythroid progenitors were P4HB, DDIA3, ARPC2 and ATP5G3. Regarding G-CSF stimulation the G-CSF receptor CSF2RB (1.1-fold) was linked via STAT3 to erythroid-specific ALAS2 (2.9-fold) and GATA2 (1.3-fold) factors, all upregulated in mPB-derived erythroid progenitors, coupled to common upregulated NUDC gene involved in the proliferation of erythroid cells. Conclusion: This report provides an extensive transcriptional profile of cultured erythroid progenitors and leads to a better understanding of diversity among the progenitor sources.

To evaluate whether the response of hematopoietic cells to interleukin-17 (IL-17) depends on the tissue microenvironment in which hematopoiesis occurs, the influence of recombinant mouse IL-17 on spleen hematopoietic cells and cytokine release was assessed in normal mice in vitro and in vivo. In vitro, IL-17 did not significantly affect the growth of granulocyte-macrophage (CFU-GM) and erythroid (BFU-E and CFU-E) derived colonies. A single injection of IL-17 in vivo exhibited stimulatory effects on hematopoietic cells from both granulocytic and erythroid lineages. The increased number of metamyelocytes 48 h after treatment imply to the IL-17-induced stimulation of granulopoiesis. The number of BFU-E was increased at 24 h, while the number of CFU-E increased 6 h and 24 h after treatment. Since the same treatment in the bone marrow decreased the number of CFU-E, it may be concluded that the local microenvironment plays an important role in IL-17-mediated effects on CFU-E. IL-17 increased the release of IL-6 both in vitro and in vivo, but showed tendency to suppress the constitutive secretion of IL-10 by spleen cells. Our results suggest the complexity of target cell response and interplay of secondary induced cytokines by IL-17 in different hematopoietic organs.

Different biological functions of hematopoietic cells are regulated by signals cells receive from their environment that may come from interactions with other cells or from soluble growth factors and cytokines. Although the effects of hematopoietic regulators are well described, the exact intracellular signaling cascades leading to various cellular responses are not fully elucidated. All hematopoietic growth factors and cytokines seem able to activate all the major signal transduction pathways simultaneously, and it appears that the same network of signaling proteins may coordinate numerous cellular functions. However, the differences that lead to the unique biological events of the particular cytokine, as well as the differences that determine lineage-specific blood cell differentiation are only gradually being uncovered. With the help of specific inhibitors of known signal transduction pathways, we have examined the contribution of particular signaling molecules of Jak/Stat, MAP kinase and PI-3 kinase pathways, as well as the activation of nuclear factor kappaB (NFkB) transcription factor on the proliferation and differentiation of murine bone marrow granulocyte-macrophage (CFU- GM) and erythroid (BFU-E and CFU-E) progenitors. Preliminary results demonstrated that the effects of the inhibitors on the hematopoietic colony formation were lineage-dependent, as well as dependent on erythroid progenitors' stage of differentiation. The obtained differences suggest that different signal transduction intermediates regulate erythroid and myeloid progenitor cell proliferation and differentiation. Differences in the susceptibility of the progenitor cells to the inhibitors used were also observed. The data is consistent with other evidences indicating that different threshold levels are one of the mechanisms by which the signaling specificity may be achieved. Mnogobrojne funkcije hematopoetskih ćelija u toku procesa hematopoeze, regulisane su brojnim signalima koje ćelije primaju iz svoje okoline i to kako od drugih ćelija tako i od brojnih regulatornih molekula. Iako su biološki efekti regulatora hematopoeze uglavnom dobro opisani, putevi prenošenja signala unutar ćelija na koje deluju nisu još uvek u potpunosti razjašnjeni. Savremena istraživanja ukazuju da gotovo svi poznati hematopoetski regulatori mogu da aktiviraju gotovo sve bitne puteve prenošenja signala unutar ćelija, što sve upućuje da ista mreža signalnih komponenti može da koordinira brojne ćelijske funkcije. Međutim, razlike u transdukciji signala koje dovode do jedinstvenog biološkog dejstva svakog citokina, kao i razlike u prenosu signala kojom se određuje lozno-zavisna diferencijacija krvnih ćelija nisu još poznati. Naša istraživanja započela su određivanjem učešća pojedinih komponenti JAK/STAT puta signalizacije, MAP kinazne kaskade i PI-3 kinaznog puta, kao i aktivacije transkripcionog faktora NFKB, u proliferaciji i diferentovanju opredeljenih matičnih ćelija granulocitno-monocitne (CFU-GM) i eritrocitne (BFU- E i CFU-E) loze kostne srži normalnih miševa. Početni rezultati ovih ispitivanja su ukazali na postojanje kako lozno-zavisnih razlika u odgovoru matičnih ćelija hematopoeze na pojedine inhibitore, tako i na zavisnost odgovora od stepena zrelosti matičnih ćelija u okviru iste loze. Dobijene razlike upućuju da su različiti učesnici signalnih puteva uključeni u regulaciju proliferacije i diferencijacije eritrocitnih i granulocitno-monocitnih progenitora. Ovo je jedan od mogućih načina kojim se možda omogućava selektivna ekspanzija ćelija određenih krvnih loza u zavisnosti od potreba organizma. Takođe, uočene su i razlike u pragu osetljivosti različitih kategorija opredeljenih matičnih ćelija hematopoeze na ispitivane inhibitore signalnih puteva, što ide u prilog podacima da su različiti pragovi osetljivosti jedan od mogućih mehanizama kojim se najverovatnije ostvaruje specifičnost delovanja signalnih puteva.

Steady-state hematopoiesis takes place in the bone marrow permissive microenvironment, composed of stromal cells, as well as extracellular matrix (ECM) components and regulatory molecules, produced by both stromal and hematopoietic cells. Stromal cells are both mezenchymal (endothelial cells fibroblasts, adipocytes, osteoblasts, myocytes, adventitial reticular cells) and hematopoietic (macrophages) in origin, which together provide cell to cell interactions, matrix proteins and growth factors essential for the maintenance, growth and differentiation of hematopoietic stem and progenitor cells. The ECM components (collagen, laminin, fibronectin, thrombospondin proteoglycans, hemonectin and various proteinases participating in their remodeling) are involved in different biological functions such as cell adhesion, binding and presentation of various cytokines and regulation of cell growth. These components serve to localize hematopoietic cells in the specific bone marrow microenvironment and it is suggested that in combination with cytokines are crucial for their compartmentalization. The regulatory molecules of hematopoietic microenvironment are cytokines, which regulate the survival, proliferation and differentiation of hematopoietic cells and cell adhesion molecules, which are responsible for the localization of hematopoiesis to the bone marrow. The activity of cytokines may be greater when the cytokine is present in a membrane-bound or ECM-associated form. Hematopoietic cells could also regulate normal hematopoiesis in an autocrine/paracrine manner, since it was shown that these cells express and secrete various growth factors, cytokines and chemokines. Proces hematopoeze se odvija u definisanoj tkivnoj mikrosredini kostne srži koja ima ključnu ulogu u njegovoj regulaciji. Mikrosredina hematopoeze podrazumeva funkcionalno jedinstvo stromalnih ćelija i ćelijskih produkata (molekuli ekstracelularnog matriksa i regulatorni faktori), koji čine kompleksni molekularni milje u kome se ostvaruju specifične interakcije hematopoetskih ćelija i komponenti mikrosredine. Stromalne ćelije mezenhimalnog porekla (endotelne ćelije, fibroblasti, adipociti osteoblasti, miociti, adventicijske ćelije retikuluma) i nemezenhimalnog porekla (makrofage), deluju na matične ćelije hematopoeze direktno među ćelijskim interakcijama, kao i produkcijom i deponovanjem pojedinih komponenti kompleksnog ekstracelularnog matriksa, a takođe i produkcijom i koncentrovanjem lokalnih citokina i faktora rasta sa hematopoetskim efektima, obezbeđujući na taj način gotovo sve činioce neophodne za proliferaciju i diferencijaciju matičnih ćelija hematopoeze. Komponente ECM (kolagen, laminin, fibronektin, trombospondin, proteoglikani, hemonektin i drugi glikoproteini kao i proteolitički enzimi koji omogućavaju remodelovanje ECM) su uključene u ćelijsku adheziju, vezivanje i prezentaciju različitih citokina i regulaciju ćelijskog rasta. Smatra se da komponente ECM omogućavaju lokalizuju hematopoetskih ćelija u specifičnoj mikrosredini kostne srži, i da u kombinaciji sa citokinima imaju presudnu ulogu u formiranju specifičnih niša. Regulatorni faktori hematopoetske mikrosredine su citokini koji regulišu preživljavanje, proliferaciju i diferencijaciju hematopoetskih ćelija i ćelijski adhezivni molekuli koji su odgovorni za lokalizaciju hematopoeze u kostnoj srži i za posredovanje u fizičkoj vezi između hematopoetskih ćelija i stromalnog tkiva mikrosredine. Ovi molekuli se u mikrosredini kostne srži nalaze kao solubilni, ili vezani za membrane stromalnih ćelija ili za komponente ECM, što može da pojača njihovu aktivnost. Takođe i same hematopoetske ćelije učestviju u regulaciji procesa hematopoeze produkujući citokine koji ispoljavaju autokrino/parakrine efekte.

The pig is widely used as a large animal model for biomedical research and could be an interesting experimental model for studies of the hematopoietic system and its response in physiological and pathological conditions. With the intention of using the pig as a large animal model in hematopoietic research, a clonal assay in methylcellulose was developed and standardized for detection of committed progenitors of the granulocyte-macrophage lineage from adult pig bone marrow and peripheral blood. Progenitor cells were stimulated to proliferate and differentiate in vitro by adding pig leukocyte conditioned medium (LCM) as a source of homologous growth factors. The number of CFU-GM (Colony Forming Unit -Granulocyte-Macrophage) directly depended on the concentration of LCM. The proliferative rate of CFU-GM progenitor cells was determined by the cytosine arabinoside suicide technique. The percentage of bone marrow and peripheral blood CFU-GM cells in S phase of the cell cycle was 34.7% and 22.2%, respectively. The data obtained regarding the number and characteristics of pig bone marrow and peripheral blood CFU-GM confirmed that the organization of the pig CFU-GM progenitor cell compartment is similar and comparable to that in miniature swine, other animal species and humans. Svinja je životinja koja se koristi kao model u različitim biomedicinskim istraživanjima, a mogia bi biti i interesantan model u ispitivanju fiziologije i patolofizioloških promena hematopoetskog sistema. U cilju razvoja eksperimentalnog modela svinje u istraživanju hematopoeze, stanadardizovan je esej za odredivanje i karakterizaciju opredeljenih matičnih ćelija granulocitno-monocitne loze iz kostne srži i krvi odrasle svinje. Stimulacija proliferacije i diferencijacije ovih matičnih ćelija postignuta je dodavanjem medijuma kondicioniranog leukocitima (LCM - Leukocyte conditioned medium) bogatog faktorima rasta. Broj CFU-GM (Colony forming unit- granulocyte-macrophage) je direktno zavisio od koncentracije LCM-a. Procenat CFU-GM ćelija u S fazi ćelijskog cikiusa odredjivan je tehnikom 'suicida' korišćenjem citozin arabinozida (Ara-C) i iznosio je 34.7% za CFU-GM iz kostne srži i 22.2% za CFU-GM iz periferne krvi. Podaci dobijeni za broj i karakteristike CFU-GM iz kostne srži i periferne krvi potvrđuju da je ovaj odeljak matičnih ćelija kod odraslih svinja organizovan na isti način kao i kod minijaturnih svinja, drugih vrsta životinja i ljudi.

The pig is widely used as a large animal model for biomedical research and could be an interesting experimental model for studies of the hematopoietic system and its response in physiological and pathological conditions. With the intention of using the pig as a large animal model in hematopoietic research, a clonal assay in methylcellulose was developed and standardized for detection of committed progenitors of the granulocyte-macrophage lineage from adult pig bone marrow and peripheral blood. Progenitor cells were stimulated to proliferate and differentiate in vitro by adding pig leukocyte conditioned medium (LCM) as a source of homologous growth factors. The number of CFU-GM (Colony Forming Unit -Granulocyte-Macrophage) directly depended on the concentration of LCM. The proliferative rate of CFU-GM progenitor cells was determined by the cytosine arabinoside suicide technique. The percentage of bone marrow and peripheral blood CFU-GM cells in S phase of the cell cycle was 34.7% and 22.2%, respectively. The data obtained regarding the number and characteristics of pig bone marrow and peripheral blood CFU-GM confirmed that the organization of the pig CFU-GM progenitor cell compartment is similar and comparable to that in miniature swine, other animal species and humans. Svinja je životinja koja se koristi kao model u različitim biomedicinskim istraživanjima, a mogia bi biti i interesantan model u ispitivanju fiziologije i patolofizioloških promena hematopoetskog sistema. U cilju razvoja eksperimentalnog modela svinje u istraživanju hematopoeze, stanadardizovan je esej za odredivanje i karakterizaciju opredeljenih matičnih ćelija granulocitno-monocitne loze iz kostne srži i krvi odrasle svinje. Stimulacija proliferacije i diferencijacije ovih matičnih ćelija postignuta je dodavanjem medijuma kondicioniranog leukocitima (LCM - Leukocyte conditioned medium) bogatog faktorima rasta. Broj CFU-GM (Colony forming unit- granulocyte-macrophage) je direktno zavisio od koncentracije LCM-a. Procenat CFU-GM ćelija u S fazi ćelijskog cikiusa odredjivan je tehnikom 'suicida' korišćenjem citozin arabinozida (Ara-C) i iznosio je 34.7% za CFU-GM iz kostne srži i 22.2% za CFU-GM iz periferne krvi. Podaci dobijeni za broj i karakteristike CFU-GM iz kostne srži i periferne krvi potvrđuju da je ovaj odeljak matičnih ćelija kod odraslih svinja organizovan na isti način kao i kod minijaturnih svinja, drugih vrsta životinja i ljudi.

The specific influence of malnutrition on the pathophysiologic changes induced by chronic alcoholism is controversial. In an attempt to determine and demarcate the effects of protein malnutrition from those produced by alcoholism and to evaluate the precise effect of alcohol per se on cytochemical and ultrastructural properties of rat polymorphonuclear neutrophil (PMN) granules, we investigated the influence of chronic protein malnutrition or chronic alcoholism alone and in combination, in rats, After a 4 month experimental period various PMN properties, such as cytochemical, morphometrical and ultrastructural, as well, as neutrophil functions were studied. It was found that the degree of damage of PMNs induced either by ethanol or protein malnutrition alone was similar whereas their combination led to worsening of all markers of PMN functional ability. Ultrastructural changes of neutrophil granules including reduction, redistribution and atypical accumulation as well as appearance of autophagic vacuoles, confirmed their alteration which was emphasised by the additive pathophysiological interaction of alcoholism and chronic hypoprotein malnutrition.

Liposomes, closed spherical structures formed by phospholipid bilayers, have been recognized as a controlled drug delivery system. We have studied the preparation and biocompatibility of liposomes based on soya phospholipids encapsulated with alpha-tocopherol. The binding efficiency of alpha-tocopherol was analysed by comparing two methods for liposome preparation: a) dry film (DFM) and b) solvent infusion method (SIM). The degree of encapsulation achieved (88-93%) suggested a high affinity of alpha-tocopherol for liposome membrane binding. The initial concentration of alpha-tocopherol had a significant effect on the degree of encapsulation, while the effect of method used was less pronounced. In general, a higher degree of encapsulation was achieved with smaller liposome size fractions. Based on an experimentally obtained size distribution function, it can be concluded that if smaller liposomes are used, SIM seems to be more efficient due to the higher content of smaller vesicles. The in vivo application of the liposomes to CBA mice confirmed the biocompatibility and nontoxicity of such preparations. The analysis of hematological parameters in peripheral blood (determination of mature blood cells with differential count) revealed that liposomes did not express cytotoxic effects on any of the parameters tested.

The multiple effects of interleukin-1 (IL-1) on hematopoietic cells are mainly documented in disturbed hematopoiesis, but its production and participation during constitutive hematopoiesis are still unproven, To assess the involvement of IL-1 in the regulation of steady-state hematopoiesis in vivo, we have investigated the consequences of IL-1 receptor blockade by recombinant human IL-1 receptor antagonist (rhIL-1Ra) in normal CBA/H mice treated with two i.p. injections of rhIL-1Ra (2 x 50 mu g/mouse) seventeen and two hours before sacrifice. The cellularity, the number of granulocyte-macrophage (CFU-GM), the number of erythroid (BFU-E) progenitor cells and the percentage of these cells in S phase of the cell cycle, as well as the morphologically recognizable cells in bone marrow were estimated, In peripheral blood, hematocrit, the number and differential count of nucleated cells, the number of erythrocytes and the percentage of reticulocytes were determined, IL-1Ra treatment significantly reduced the number of femoral CFU-GM and BFU-E, while all the other analyzed parameters were not different from the level obtained in control, non-treated animals. These findings show that a number of bone marrow IL-l-responsive cells were affected by the IL-1 receptor blockade, indicating that the expression of IL-1 receptors and endogenous IL-1 secretion occur as part of constitutive hematopoiesis.

Recent studies have shown that the T cell-derived cytokine, interleukin-17 (IL-17), stimulates hematopoiesis, specifically granulopoiesis inducing expansion of committed and immature progenitors in bone marrow. Our previous results pointed to its role in erythropoiesis too, demonstrating significant stimulation of BFU-E and suppression of CFU-E growth in the bone marrow from normal mice. As different sensitivities of erythroid and myeloid progenitor cells to nitric oxide (NO) were found, we considered the possibility that the observed effects of IL-17 were mediated by NO. The effects of recombinant mouse IL-17, NO donor (sodium nitroprusside - SNP) and two NO synthases inhibitors (L-NAME and aminoguanidine) on erythroid progenitor cells growth, as well as the ability of IL-17 to induce nitric oxide production in murine bone marrow cells, were examined. In addition, we tested whether the inhibition of CFU-E colony formation by IL-17 could be corrected by erythropoietin (Epo), the principal regulator of erythropoiesis. We demonstrated that IL-17 can stimulate low level production of NO in murine bone marrow cells. Exogenously added NO inhibited CFU-E colony formation, whereas both L-NAME and aminoguanidine reversed the CFU-E suppression by IL-17 in a dose-dependent manner. The inhibition of CFU-E by IL-17 was also corrected by exposure to higher levels of Epo. The data obtained demonstrated that at least some of the IL-17 effects in bone marrow related to the inhibition of CFU-E, were mediated by NO generation. The fact that Epo also overcomes the inhibitory effect of IL-17 on CFU-E suggests the need for further research on their mutual relationship and co-signalling.